Benzimidazole-Triazole Hybrids as Antimicrobial and Antiviral Agents: A Systematic Review

Two series of new hybrids, 2-[4-((1H-benzimidazol-2-ylthio)methyl)-1H-1,2,3-triazol -1-yl]N′-(arylmethylidene)acetohydrazides (2a–2l) and 2-[4-((1H-benzimidazol-2-ylthio) methyl)-1H-1,2,3-triazol-1-yl]N-(α-arylethylidene)acetohydrazides (3a–3f) were prepared by Youssif et al. in two steps starting from 2-[4-((1H-benzimidazol-2-ylthio) methyl)-1H-1,2,3-triazol-1-yl] acetohydrazide 1 (Scheme 1). Compounds 2a–2l exhibited pronounced antibacterial activity, which ranged from 35 to 75% of that of the standard drug against Staphylococcus aureus and 50–80% of that of Ciprofloxacin against E. Coli (MIC values of 3.125–12.5 μmol mL⁻¹). Compound 2k showed the highest activity against S. aureus (75% activity, MIC = 12.5 μmol mL⁻¹), while compound 2d was the most active derivative against E. Coli (80% activity, MIC = 3.125 μmol mL⁻¹). All the synthesized compounds were tested as potential antifungal agents against Candida albicans using Fluconazole as a reference drug. Compound 1 showed the activity of 48% of that of Fluconazole (MIC = 12.5 μmol mL⁻¹). Compounds 2e and 2k displayed higher antifungal activity among the other derivatives as they showed 75% activity of that of Fluconazole (MIC = 3.125 μmol mL⁻¹). Compounds 3a–3f exhibited moderate to good activity against E. Coli, and their activity was 50–70% of that of Ciprofloxacin (MIC values of 6.25–12.5 μmol mL⁻¹), and compounds 3a and 3f were the most active compounds against E. coli as they showed 70% of that of Fluconazole (MIC = 6.25 μmol mL⁻¹) while compound 3b showed the highest activity against Staphylococcus aureus (65% of that of Ciprofloxacin, MIC = 18 μmol mL⁻¹) [118]. Al-blewi et al. used an azide–alkyne Huisgen cycloaddition reaction carried out by simultaneously mixing thiopropargylated benzimidazole 4 with the appropriate sulfa drug azides 5a–5f, copper sulfate, and sodium ascorbate in DMSO/H₂O to regioselectively furnish target mono-1,4-disubstituted-1,2,3-triazole tethered benzimidazole-sulfonamide conjugates 6a–6f with 85–90% yields after 6–8 h of heating at 80 °C (Scheme 2). All compounds were evaluated for their antimicrobial activity (Table 1) against four pathogenic bacterial strains (Gram-positive: Bacillus cereus ATTC 10876, Staphylococcus aureus ATTC 25,923, and Gram-negative: Escherichia coli ATTC 25922, Pseudomonas aeruginosa ATTC 27,853 and two fungal strains, Candida albicans ATTC 50193, Aspergillus brasiliensis ATTC 16404). As can be seen in Table 1, compound 6a showed the best antibacterial activity against Bacillus cereus and Staphylococcus aureus (64 μg mL⁻¹), and compounds 6c, 6d, and 6e showed the best antibacterial activity against Escherichia coli (64 μg mL⁻¹) [119]. Evaluation of in silico physicochemical properties or ADMET (adsorption, distribution, metabolism, excretion, and toxicity) as a robust tool to confirm the potential of a drug candidate was applied for these compounds [120]. As per Lipinski’s rule of five, an orally administered drug should have a log p ≤ 5, a molecular weight (MW) < 500 Daltons, and an HBD ≤ 5 [121] to be in the acceptable range. Results have shown that all hybrids have in good agreement in terms of HBD. Rashdan et al. synthesized hybrids 10 starting from 2-azido-1H-benzo[d]imidazole derivatives 7a–7b, which reacted with acetylacetone in the presence of sodium ethoxide to obtain hybrids molecules 8a–8b. The latter acted as a key molecule for the synthesis of new carbazone derivatives 9a–9b that were submitted to react with 2-oxo-N-phenyl-2 (phenylamino)acetohydrazonoyl chloride to obtain the target hybrid derivatives 10a–10b (Scheme 3). All compounds were screened for their in vitro antimicrobial activity against pathogenic microorganisms Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Aspergillus niger, and Candida albicans. The results showed that compounds 10a and 10b had strong activity against all the tested pathogenic microbes. Compounds 8a and 9a only showed effects against the Gram-negative and Gram-positive bacteria and had no effect on the tested fungi. In addition, in silico, and in vitro findings showed that compounds 10a and 10b were the most active against bacterial strains and could serve as potential antimicrobial agents (Table 2). The hybrids 8–10 were subjected to molecular docking studies with DNA gyrase B and exhibited binding energy that extended from -9.8 to -6.4 kcal/mol, which confirmed their excellent potency. The compounds 10a and 10b were found to be with the minimum binding energy (−9.8 and −9.7 kcal/mol) as compared to the standard drug Ciprofloxacin (−7.4 kcal/mol) against the target enzyme DNA gyrase B, as summarized in Figure 2 [122].

ADMET analysis of compounds 8–10 exhibited that they have good absorption properties (%HIA) ranging from 99.57 to 100% [123]. For distribution, the compounds do not permeate the blood–brain barrier (BBB). Moreover, the molecules were negative in the AMES toxicity and carcinogenicity test, which suggests that they are non-mutagenic. Compounds 11a–11g with terminal acetylene and 2-(azidomethoxy)ethyl acetate were condensed using CuI as catalyst and triethylamine (TEA) under microwave irradiation to achieve hybrids 1,2,3-triazole connected via benzene to the benzimidazole nucleus 12a–12g with excellent yields (70–90%) (Scheme 2). The cleavage of the acetyl group using potassium carbonate (K₂CO₃) in methanol liberated the hydroxy group of the corresponding hybrid triazoles 13a–13g (Scheme 4). in almost quantitative yields. Compounds 13a–13g were screened for in vitro antifungal activities against two phytopathogenic fungi, Verticillium dahliae Kleb and Fusarium oxysporum f. sp. albedinis. The result of the mycelia linear growth rate indicates that some of the compounds show a weak inhibition against the two fungi, the only compound that shows a significantly increased rate is compound 13e, with a rate of 29.76% against Verticillium dahliae in the sporulation test [124].

Bistrović et al. synthesized in two steps hybrids 19a–19e, 20a–20e, and 21a–21e starting from 4-(prop-2-ynyloxy)benzaldehyde 14 (Scheme 5). All compounds were evaluated for their in vitro antibacterial activity against Gram-positive bacteria: S. aureus ATCC 25923, methicillin-sensitive S. aureus, E. faecalis, vancomycin-resistant E. faecium, and Gram-negative bacteria: E. coli ATCC 25925, P. aeruginosa ATCC 27853, A. baumannii ATCC 19,606 and ESBL-producing K. pneumoniae ATCC 27736. Generally, compounds showed better activities against Gram-positive than Gram-negative bacteria. Compounds 20a–20e, with better binding affinity relative to other amidines, were the most active against S. aureus (MIC = 8–32 µgmL⁻¹). Compound 19a was the most promising candidate because of its higher potency (MIC = 4 µgmL⁻¹) against ESBL-producing E. coli. Results of anti-trypanosomal evaluations showed that p-methoxyphenyl substituent in 19b–21b enhanced activity, with 20b (IC₅₀ = 1.1 mM and IC₉₀ = 3.5 mM) being more potent than Nifurtimox. In contrast to the observed correlation between antimicrobial activity and DNA binding, the antiprotozoal effects of 20b did not correlate with its DNA affinity [125].

Rao et al. synthesized hybrids 22a–22b (Figure 3) using the click chemistry approach. Compounds had weak activity against Mycobacterium bovis strain, with BCG values % inhibition = 27.3 and 26.2, respectively, at 30 µM concentration, using a turbidometric assay. Compounds 22a and 22b also showed moderate antiproliferative activity against human breast cancer cell line MCF-7, with IC₅₀ values of 31.9 and 25.1 μM, respectively [126]. Ashok et al. synthesized in three steps hybrids 26a–26j, starting from 1H-indole-3-carbaldehyde 7 (Scheme 6). The compounds were evaluated for their antimicrobial activity against Gram-positive Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, and Gram-negative Proteus vulgaris ATCC 29213, Escherichia coli ATCC 11,229 bacteria using Gentamicin as standard. Antifungal activity was tested against Candida albicans ATCC 10,231 and Aspergillus niger ATCC 9029 strains with the standard drug Fluconazole. Compounds 26b, 26c, and 26h with MIC of 3.125–6.25 μg mL⁻¹ were found to be the most promising potential antimicrobial molecules [127]. The authors calculated various physicochemical parameters such as clogP, drug score, and drug-likeness of 26a–26j using the Osiris Property Explorer software [128]. For all the compounds, the calculated clogP values were found to be below five according to Lipinski’s rule-of-5 and also exhibited positive values for drug score. Mallikanti et al. synthesized novel benzimidazole-conjugated 1,2,3-triazole analogs 29a–29l in two steps: 1. formation of benzimidazole intermediate by reaction between 3’,5’-difluorobiphenyl-3,4-diamine 27 and 2-hydroxy-4-(prop-2-ynyloxy) benzaldehyde 28, and 2. microwave-assisted copper-catalyzed click reaction (Scheme 7). Compounds 29a–29l showed minimal inhibition zones against all Gram-positive (S. aureus, B. subtilis) and Gram-negative (E. coli, P. aeruginosa) strains using Ampicillin as A standard drug. Among all tested compounds, the 29i and 29k showed higher activity against P. aeruginosa, S. aureus, and B. subtilis than the standard reference. Compounds 29a, 29b, 29c, 29d, 29e, 29f, 29g, 29h, 29j, and 29l showed moderate antibacterial activity against tested strains (Inhibition zone: 10–25 mm compared with 18–20 mm for Ampicillin). Compounds 29i, 29j, and 29k also established strong activity against both fungal strains, C. albicans MTCC 183 and A. niger MTCC 9652, compared to the standard drug Griseofulvin [70]. To understand the binding mode of novel compounds, docking simulations were performed against the crystal structures of glucosamine-6-phosphate synthase (GlmS) (PDB ID: 2VF5) of E. coli and secreted aspartic proteinase (Sap) 1 (PDB ID: 2QZW) of C. albicans retrieved from the protein data bank. The best active compound, 29l, scored the highest binding affinity value of about −10.0 kal/mol, which demonstrated two key interactions with the active site amino acid Asp549 of Glms with a bond distance of 2.66 and 2.81 Å. Further, the hydrophobic interactions were taken with Tyr312, Ser316, Asp474, Asn523, Ala572, and Ala551 of Glms, among which one π-π T-shaped interaction with Tyr312, and halogen bond [129] interactions with Tyr312, Asn523, and Asn551 (Figure 4). The binding energies and interactions of all compounds are better than that of Ampicillin, which proves that these molecules could best fit into the cavity of Glms [70]. Chandrika et al. reported hybrids 30–32 with broad spectrum antifungal activity (0.975–3.9 µgmL⁻¹ against C. albicans; 0.12–0.48 µgmL⁻¹ against C. parapsilosis) (Figure 5). These compounds also displayed good activity against C. albicans biofilms (3.9–15.6 µgmL⁻¹ against C. albicans) [130].

Deswal et al. synthesized a new series of benzimidazole-1,2,3-triazole-indoline derivatives 35 by employing a click reaction between substituted N-propargylated benzimidazole derivatives 33 and in situ formed substituted 2-azido-1-(indolin-1-yl) ethanone derivatives 34, in moderate to good yields (Scheme 8). The obtained results indicate a stronger inhibitory effect of compound 35d against E. coli, while compound 35g showed good inhibition against all the tested strains except B. subtilis (Table 3). The good antimicrobial activity of the compounds was correlated with the presence of the pyridine ring in position ‘‘2’’ of the benzimidazole and the NO₂ group on the indole ring. Furthermore, in vitro α-glucosidase inhibition of all synthesized derivatives identified 35e (IC₅₀ = 0.015 ± 0.0003 μmol mL⁻¹) and 35g (IC₅₀ = 0.018 ± 0.0008 μmol mL⁻¹) as potent inhibitors of α-glucosidase, even better than standard drug Acarbose [6].

Saber et al. synthesized new 1,4-disubstituted-1,2,3-triazole containing benzimidazolone derivatives 37a–37d exclusively using click chemistry (Scheme 9). All derivatives exhibited antibacterial activity against tested strains, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, but compounds 37b and 37d are more effective against Gram-positive bacterium S. aureus (MIC = 3.125 µgmL⁻¹), and 37b has better activity against Gram-negative bacterium E. coli (MIC = 3.125 µgmL⁻¹) with Chloramphenicol as standard drug. The expected inhibition efficiency, 37c > 37a > 37b, was attributed to the favorable effect of the side carbon chain of the triazole moiety, according to DFT calculations, in this process [131,132]. Mohsen et al. synthesized hybrids 41a–41e in three steps, starting from benzimidazole 38, namely two alkylation reactions and a click reaction (Scheme 10). New derivatives exhibited good zone inhibition of 6.8, 5.4, 5.2, 4.5, and 5.3 mm for the S. aureus strain and 5.4, 3.8, 4.2, 3.3, 4.9 mm for the E. coli strain, indicating that the 1,2,3-triazole core contributed significantly to bacterial growth suppression (Ciprofloxacin showed 10.2 mm for S. aureus and 10.4 mm for E. coli). Compared with Gram-negative bacteria, all compounds showed a strong effect against Gram-positive bacteria [94].

Rezki reported the intramolecular cyclization of thiosemicarbazides 42a–42d in refluxing aqueous sodium hydroxide (2N) for 6 h with the formation of hybrids 43a–43d with yields of 82–86% (Scheme 11). Among all the 1,2,4-triazole derivatives, N4-phenyl and N4-(4-fluorophenyl) derivatives 43a and 43b were found to be the most potent with MIC values of 4–8 µg mL⁻¹. Also, triazoles 43c and 43d exerted the best inhibition against both tested fungal strains, A. brasiliensis and Candida albicans, with MIC values ranging from 0.5 to 4 µg mL⁻¹, more potent than the reference drug Fluconazole. Condensation of compound 44 with several benzaldehydes in refluxing ethanol for 4–6 h with a catalytic amount of HCl produced a new class of hybrid Schiff bases 45a–45g with yields of 84–86% (Scheme 12). The antimicrobial bioassay results for the synthesized Schiff bases 45a–45g revealed that all of the tested compounds were more effective towards all of the organisms, with MIC values of 1–16 µg mL⁻¹. Among them, Schiff bases 45c, 45d, and 45e with a fluorine atom at position “2” exhibited the highest antibacterial inhibition potency at MIC 1–8 µg mL⁻¹. The Schiff base 45e containing a CF₃ group exerted the highest antifungal inhibition activity with MIC of 1 µg mL⁻¹ [133]. Al-blewi et al. synthesized triazoles 47a–47f in two steps: i. regioselective alkylation of 4 with two equivalents of propargyl bromide in the presence of two equivalents of potassium carbonate as a base catalyst to afford benzimidazole 46 with 91% yield after stirring at room temperature overnight; ii. Copper-mediated Huisgen 1,3-dipolar cycloaddition reaction on compound 46 in good yields (82–88%) (Scheme 13). Generally, bis-1,2,3-triazoles 47a–47f exhibited more potent antimicrobial activities than their mono-1,2,3-triazole derivatives 6a–6f. This was attributed to the synergistic effect of the sulfonamoyl and tethered heterocyclic components in addition to the improved lipophilicity of the bis-substituted derivatives. Among the synthesized compounds, compound 47a was the most potent antimicrobial agent, with MIC values ranging between 32 and 64 μg mL⁻¹ against all tested strains B. cereus, S. aureus, E. coli P. aeruginosa, C. albicans, and A. brasiliensis. Pharmacophore elucidation of the compound 47a–47f was performed based on in silico ADMET evaluation of the tested compounds. Screening results of drug-likeness rules showed that all compounds follow the accepted rules, meet the criteria of drug-likeness, and follow Lipinski’s rule of five. In addition, the toxicity results showed that all compounds are non-mutagenic and noncarcinogenic [119].

Aparna et al. used a similar strategy for obtaining nine new bis-1,2,3-triazol-1H- 4-yl-substituted arylbenzimidazole-2-thiol derivatives 48a–48l (Figure 6). Antibacterial activity of triazole derivatives 48 demonstrates moderate to good activity against Gram-negative (E. coli, S. typhy, P. aeruginosa) and Gram-positive (S. aureus) bacterial strains. The products 48i, 48k, and 48l are characterized by a broad spectrum of antibacterial activity at a concentration of 10 μg mL⁻¹. The synthesized 1,2,3 triazole derivatives were studied for their molecular docking on the high-resolution X-ray crystal structure of FabI of Staphylococcus aureus (pdb id:4FS3) obtained from the protein data bank [134]. The highest dock score of –7.69 kcal/mol and the lowest dock score of –0.942 kcal/mol were obtained for molecules 48l and 48h, respectively [135].

Ashok reported compound 26h has the best antitubercular drug candidate by inhibiting the growth of the MTB (Mycobacterium tuberculosis) strain with MIC = 3.125 μ mL⁻¹ (7.1 μM) (control Rifampicin MIC = 0.04 μg mL⁻¹ and isoniazid MIC = 0.38 μg mL⁻¹). The best antitubercular activity of 26h may be attributed to the presence of the nitro group on the phenyl ring at the ortho position. Compound 26b (MIC = 6.25 μg mL⁻¹ (14.7 μM)) with chlorine substituent, compound 26i (MIC = 6.25 μg mL⁻¹(14.2 μM)) with trifluoromethyl substituent and compound 26j (MIC = 12.5 μg mL⁻¹ (28.4 μM)) with benzyl substituent exhibited moderate antitubercular activity. Therefore, the incorporation of the electron-withdrawing nitro group, electronegative chlorine, and trifluoromethyl groups on the phenyl ring was highly favored for antitubercular activity. The authors calculated various physicochemical parameters and found from the theoretical data that compounds 26a–26j also exhibited positive values for drug score [127]. Gill et al. reported syntheses of hybrids 51a–51d by reaction between 2-(3-fluorophenyl)-1H-benzo[d]imidazole 50 and phenyl-substituted 4-(bromomethyl)-1-phenyl-1H-1,2,3-triazole 49 in DMF at room temperature (Scheme 14). Trifluorosubstituted-compound 51a possessed enhanced anti-mycobacterial activity, >96% of inhibition at 6.25 µg concentration. Also, compounds 51b and 51c, which had antimicrobial activities superior to the other compounds, were reported as the best choice for the preparation of new derivatives in order to improve effectiveness on intracellular mycobacteria (macrophage) or in infected animals [136]. Anand et al. reported a one-pot reaction between 2-propargylthiobenzimidazole 4, 4-bromomethylcoumarins/1-aza-coumarins 52/53 and sodium azide under click chemistry conditions to give exclusively 1,4-disubstituted triazoles 54a–54n. (Scheme 15). Antitubercular assays against M. tuberculosis (H37Rv) coupled with in silico molecular docking studies indicated that dimethyl substituents 54c and 54d showed promising activity (MIC = 3.8 µMol L⁻¹) with higher C-score values. Surflex-Dock was used to investigate detailed intermolecular interactions between the ligand and the target protein. Three-dimensional structure information on the target protein was taken from the PDB entry 4FDO. Processing of the protein included the removal of the co-crystallized ligand and water molecules, as well as the addition of essential hydrogen atoms. All 14 inhibitors 54a–54n were docked into the active site of ENR, as shown in Figure 7a, and Figure 7b indicates the superimposition of compounds 54a and 54d with ligand [137]. Khanapurmath et al. synthesized triazoles 55 by click reaction (Figure 8a). Benzimidazolone bis-triazoles 55a–55n showed better activity with MIC in the range 2.33–18.34 μM, and the most active compounds were 55h and 55m. All compounds exhibited moderate to low levels of cytotoxicity with IC₅₀ values of the human embryonic kidney cells in the range of 943–12294 μM, and none of the 14 compounds exhibited any significant cytotoxic effects, suggesting huge potential for their in vivo use as antitubercular agents. Docking studies revealed an additional interaction of benzimidazolone oxygen in these compounds (Figure 8b) [138]. Also, Sharma et al. summarize 1,2,3-triazoles as antitubercular compounds and various hybrids with benzimidazole, coumarin, isoniazid, quinolines, etc. [39].

Pandey et al. synthesized hybrids 59a–59e in three steps: reaction of 7-hydroxy-4-methyl coumarin with thiosemicarbazide to form triazole intermediate 57, which underwent Mannich reaction with formaldehyde, and an amino acid to form intermediates 58a–58e. Intermediates 58a–58e reacted with o-phenylenediamine in pyridine to give benzimidazole-1,2,4-triazole hybrids in poor yields (Scheme 16). Compound 59a displayed promising antifungal activity against Candida albicans and Cryptococcus himalayensis since the MIC value in each case was found to be 3.5 μg mL^–1. Compound 59b showed low to moderate antifungal activity against all five fungi, Candida albicans, Cryptococcus himalayensis, Sporotrichum schenkii, Trichophyton rubrum, and Aspergillus fumigatus [139].

Jadhav et al. synthesized a series of hybrids 1,2,4-triazolyl-fluorobenzimidazoles in two steps: i. synthesis of 2-(4-(1H-1,2,4-triazol-1-yl)phenyl)-4,6-difluoro-1H-benzo [d]imidazole 62 by reaction between 3,5-difluorobenzene-1,2-diamine 60 and 4-(1H-1,2,4- triazol-1-yl)benzaldehyde 61 in toluene at 110 °C, and ii. alkylation of compound 62 in DMF at room temperature, with the formation of the final hybrids 63a–63o (Scheme 17). All compounds were screened for antimicrobial activity against different Gram-positive organisms, S. aureus, P. aeruginosa, and Gram-negative organisms, E. coli and S. typhosa using Gentamycin as a reference standard. The data generated from preliminary screening showed that compounds displayed moderate to better antimicrobial activity. Compounds 63a, 63e, 63f, 63h, 63i, and 63l displayed maximum activity (Table 4) [140].

Barot et al. synthesized hybrid 64 and determined its antimicrobial activity against Bacillus cereus MTCC-430, Enterococcus faecalis MTCC-493, S. aureus MTCC-737, Escherichia coli MTCC-1687, Pseudomonas aeruginosa MTCC-2642, Klebsiella pneumonia MTCC-109, Candida albicans MTCC-3017, Aspergillus niger MTCC-1344 and Fusarium oxyspora MTCC-1755, of MIC = 13–18 µg ml⁻¹, with Ofloxacine and Fluconazole as standard drugs [141]. Also, Jiang et al. reported antifungal activity for hybrid 65 against Candida albicans, Candida tropicalis, Cryptococcus neoformans, Trichophyton rubrum, and Aspergillus fumigatus of MIC₈₀ = 1–64 µg mL⁻¹ considering Fluconazole as a standard drug (Figure 9). From the antifungal activity data, preliminary SARs was obtained. In general, the amine linker was important for antifungal activities. Substituted piperazine derivatives were comparable or superior to the corresponding N-methyl derivatives. [142]. Luo et al. reported a series of naphthalimide benzimidazole-1,2,4-triazole hybrids 68a–68h and the corresponding triazolium salts 69a–69d prepared by convenient and efficient procedures starting from naphthalimide triazole 66 (Scheme 18). 2-Chlorobenzyl triazolium 68g and compound 69b with octyl group exhibited the best antibacterial activities among all the tested compounds, especially against S. aureus with an inhibitory concentration of 2 μg mL⁻¹ which was equipotent potency to Norfloxacin (MIC = 2 μg mL⁻¹) and more active than Chloromycin (MIC = 7 μg mL⁻¹). Triazoliums 68g and 68f bearing 3-fluorobenzyl moiety displayed the best antifungal activities (MIC = 2–19 μg mL⁻¹) against all the tested fungal strains, C. albicans ATCC 76615, A. fumigatus ATCC 96918, C. utilis, S. cerevisia and A. flavus, without being toxic to PC12 cell line within concentration of 128 μg mL⁻¹. Further investigations showed that compound 68g could intercalate into calf thymus DNA to form the 68g-DNA complex, which could block DNA replication, exerting powerful antimicrobial activities. [143]. Benzimidazole-1,2,4-triazole Mannich base 70 was active against Bacillus subtilis and Bacillus pumilus (inhibition zone diameters being 19 and 17 mm, respectively, compared to Ciprofloxacin with 28 and 30 mm, respectively) [144].

Kankate et al. reported the synthesis of hybrids 73a–73l (Scheme 19). The antifungal activity of compound 73 was tested against Candida albicans spores in vitro (turbidimetric method) and in vivo (kidney burden test). Compound 73i had a good antifungal activity as compared with the other twelve compounds at 0.0075 µM mL⁻¹, which is equivalent to Fluconazole activity both in vitro and in vivo. The antifungal activity decreased with the increasing alkyl length of N1 of benzimidazole (methyl to ethyl). This was proven for compounds 73i and 73l, which showed MICs of 0.0075 and 0.015 µM mL⁻¹, respectively. The ligand fit method was performed to study and predict the binding mode of the hybrids 73 with the target enzyme (homology modeled) cytochrome P450 lanosterol 14-α-demethylase of C. albicans. All compounds showed binding in the active site of the enzyme. The 1,2,4-triazole ring of compounds 73a–73l is positioned almost perpendicular to the porphyrin plane, with a ring nitrogen (N-4) atom coordinated to the heme iron (Figure 10) [145,146]. Ahuja et al. reported antifungal activity of compounds 74a–74c against F. verticillioides, D. oryzae, C. lunata, and F. fujikuroi (Figure 11). All compounds had increased potency than the standard commercial benzimidazole fungicide, carbendazim (Table 5).

Compound 74c exhibited ED₅₀ values lower than triazole fungicide, propiconazole. The results reinforced the synergistic effects of the benzimidazole and 1,2,4-triazole combination supported by a computational approach. Hydrogen bonding interactions were more pronounced in compounds 74a–74c in the binding pockets of both the target enzymes, in comparison to standards. In compound 74c, two H-bonds were formed with Gln11 present in the binding cleft of the active pocket of β-tubulin (Figure 12). In all three compounds, the right position of the N-atoms of both 1,2,4-triazole and benzimidazole, that O-atoms of methoxy and carbonyl groups, contributed well to strong binding into the active site of enzymes via H-bonding [147]. Evren et al. reported the synthesis of the compounds 79a–79c in two steps: i. reaction of 1,2,4-triazole 75 with 4-fluorobenzaldehyde 76 in DMF with the formation of 4-(1H-1,2,4-triazol-1-yl)benzaldehyde 77; ii. reaction of aldehyde 77 with 1,2-phenylene diamines 78 (Scheme 20). Although the antibacterial activities of compounds 79a–79c against Escherichia coli ATCC 35218, E. coli ATCC 25922, Klebsiella pneumoniae NCTC 9633, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 13311, and Staphylococcus aureus ATCC 25923, were weak, the antifungal activities against C. albicans were found promising, with MIC values of 3.9, 7.8, and 3.9 μg mL⁻¹ respectively, using as reference drug Ketokonazole (MIC = 7.8 μg mL ⁻¹). Protein-ligand interactions and binding poses of the 79a, 79b, and 79c compounds on the CYP51 active site were examined. As shown in Figure 13, an H bond of 2.11 Å between compound 79a and Met508, π-π stacking interactions with Tyr118, Hie377, Phe233, generated hydrophobic interactions with Pro230, Leu376, Tyr64, Phe228, and Tyr505. Theoretical ADME calculations of the 79a, 79b, and 79c were made, and the compounds were found to have good lipophilicity, moderate water solubility, and within the limiting rules of Lipinski, Ghose, Veber, Egan, and Muegge (Figure 14) [148]. Ghobadi et al. reported the synthesis of compounds 85a–85e, in two different ways, from 3,4-diaminobenzophenone 80, i. formation of 2-mercapto benzimidazole derivatives 82, 83, and ii. nucleophilic ring opening of various oxiranes 84a–84e with benzimidazoles 82 and 83 using NaHCO₃ in ethanol at room temperature (Scheme 21). Compounds 85a–85e, containing a 5-benzoylbenzimidazole scaffold, showed better antifungal activity against Candida spp. and Cryptococcus neoformans than related benzimidazole and benzothiazole derivatives. The better results were obtained with 4-chloro-derivative 85b displaying MICs < 0.063–1 μg mL⁻¹. Also, compound 86c, synthesized analogously, is as potent as compound 85b. The docking experiments were conducted to further rationalize the obtained antifungal activity data and investigate the type of interactions between compound 85b and the active site of lanosterol 14α-demethylase (CYP51). As shown in Figure 13, the coordinated bond-forming distance between the N4 atom of the triazole nucleus of compound 85b and the iron atom in the heme group of active site were 2.71 and 2.40 Å, respectively. A hydrogen-bonding interaction between Tyr132 and the sulfur group of (S)-85b was observed. In vitro and in silico ADMET evaluations of the most promising compounds 85b indicated that the selected compounds have desirable ADMET properties in comparison to the standard drug Fluconazole. A docking simulation study demonstrated that the benzimidazol-2-yl-thio moiety is responsible for the potent antifungal activity of these compounds [72].

Ansari et al. synthesized hybrids 88a–88c in two steps from 2-(2-methyl-1H-benzo [d]imidazol-1-yl)acetohydrazide 87 (Scheme 22). Generally, all benzimidazole-triazole hybrids showed low antimicrobial activity (Table 6) [149]. Tien et al. synthesized hybrids 89a–89d in three steps from 2-(2-methyl-1H-benzo[d]imidazol-1-yl)acetohydrazide 87b (Scheme 23). All compounds exhibited antifungal activity against A. niger (MIC = 50 µg mL⁻¹). Only compound 89b exhibited activity against F. oxysporum (Table 7) [150]. Kantar et al. reported antimicrobial activity of hybrid 90 (Figure 15) against four Gram-positive, Bacillus cereus 702 Roma (62.5 µg mL⁻¹), B. megaterium DSM-32 (125 µg mL⁻¹), B. subtilis ATCC 6633 (62.5 µg mL⁻¹), Staphylococcus aureus ATCC 25,923 (250 µg mL⁻¹), and four Gram-negative bacteria, Escherichia coli ATCC 25,922 (250 µg mL⁻¹), Enterobacter cloaceae ATCC13047 (125 µg mL⁻¹), Pseudomonas aeruginosa ATCC 27,853 (250 µg mL⁻¹), and Yersinia pseudotuberculosis ATCC 911 (125 µg mL⁻¹) bacteria [151]. Nandwana et al. reported compound 91 synthesized in good yield (70%) with promising antibacterial activity, with minimum inhibitory concentration (MIC) values of 4−8 μg mL⁻¹ for all bacterial tested strains (Escherichia coli, Pseudomonas putida, Salmonella typhi, Bacillus subtilis, Staphylococcus aureus), as compared to the positive control Ciprofloxacin, and also with pronounced antifungal activity against both tested strains, Aspergillus niger and Candida albicans (MIC = 8−16 μg mL⁻¹) as compared with Amphotericin B [152]. Al-Majidi et al. synthesized 2-mercaptobenzimidazole derivatives 95, 96, and 97 by cyclization of intermediate precursors 93, 94, and 95 under reflux with 2N NaOH (Scheme 24). The compounds generally showed moderate antimicrobial activity against all tested strains, as can be seen in Table 8 [153]. El-masry et al. synthesized compounds 98 and 99 and found that they did not exhibit antimicrobial activity (Figure 16) [154]. Menteşe et al. synthesized compounds 100a–100d, for which they found no antimicrobial activity on the ten strains tested [155]. Karale et al. synthesized bis-benzimidazole-1,2,4-triazole hybrids 102a–102e (Scheme 25) in four steps from 7-methyl-2-propyl-3H-benzo[d]imidazole-5-carboxylic acid. All compounds 102 did not show antimicrobial activity against the strains tested, C. albicans, A. fumigatus, S. aureus, and E. coli [156,157].

Eisa et al. synthesized compounds 105a and 105b (Scheme 26) (Table 9) by the reaction between 2-(chloromethyl)-1H-benzo[d]imidazole 103 and 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4- triazole-3-thiol 104a or 4-phenyl -5-(thiophen-2-yl)-4H-1,2,4-triazole-3-thiol 104b, at reflux in absolute ethanol, for 12 h. Also, they reported synthesis of the compounds 107a and 107b from 2-(2-(phenylthiomethyl)-1H-benzo[d]imidazol-1-yl)acetohydrazide in two steps (Scheme 27). All compounds showed antimicrobial activity against Escherichia coli superior to that of standard Gentamicin. Compound 107a exhibited only moderate activity against Staphylococcus aureus [158]. Nevade et al. synthesized compounds 109a–109h in five steps from 1H-benzo[d]imidazole-2-thiol 108 (Scheme 28). The antimicrobial screening results presented in Table 10 reveal that compounds 109a, 109c, and 109e exhibited satisfactory effects against S.aureus and E.coli, while compounds 109b, 109f, and 109g showed moderate activity against the same microbes. Also, the antifungal activity of these compounds was screened against Candida albicans. Compounds 109a and 109d showed the highest degree of inhibition against C.albicans when compared with the standard drug Ketoconazole [159]. Can et al. synthesized hybrids 111a–111h in four steps from methyl 4-(5-methyl-1H-benzo[d]imidazol-2-yl)benzoate 110 (Scheme 29). All compounds were screened for antifungal activity against Candida albicans ATCC 24433, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22,019 (Table 11). Compounds 111i and 111s exhibited significant inhibitory activity against Candida strains with MIC₅₀ values ranging from 0.78 to 1.56 μg mL⁻¹ [160]. Gencer et al. synthesized compounds 112 in good yields (77–88%) using a similar strategy (Figure 17). Microbiological studies revealed that compounds 112a, 112b, 112c, 112e, 112f, 112g, and 112h possess a good antifungal profile against all tested strains, C. albicans, C. glabrata, C. krusei, C. parapsilopsis, with MIC₅₀ = 0.78–1.56 µg mL⁻¹. Compound 112i was the most active derivative and showed comparable antifungal activity to those of reference drugs Ketoconazole and Fluconazole [161]. The SAR (Structure–activity relationship) on the synthesized benzimidazole-triazole compounds is summarized in Figure 18. It is observed that the presence of chlorine or fluorine in the “5” position of benzimidazole, as well as the presence of fluorine in the “4” position of phenyl, increase the antibacterial activity, while the presence of fluorine in the “2” position of phenyl does not change the activity, and the presence of groups CH₃ or C₂H₅ in position “4” in the triazole nucleus does not bring any change in the antibacterial activity of the compounds. Furthermore, toxicological and ADME studies indicated the relative potency of hybrids 112h and 112i, according to the literature [162,163,164,165,166]. Compound 112i also inhibited ergosterol biosynthesis concentration dependently. Results of ergosterol level quantification assay and fluorescence microscopy studies revealed that the mechanism of action of hybrids is associated with the inhibition of ergosterol biosynthesis, which may subsequently result in altered membrane fluidity, plasma membrane biogenesis, and functions of fungi. Güzel et al. synthesized a new series of benzimidazole-1,2,4-triazole derivatives 113a–113l using the same procedure described in Scheme 29 as potential antifungal agents (Figure 19). All the compounds were screened for their in vitro antifungal activity against four fungal strains, namely, C. albicans, C. glabrata, C. krusei, and C. parapsilopsis and were found to exhibit excellent activity against C. glabrata. Especially, compounds 113b, 113i, and 113j were found to be the most effective compounds in the series with an MIC value of 0.97 μg mL⁻¹ [71]. According to the molecular docking study, compounds 113b, 113i, and 113j fit into the LDM enzyme active pocket. In a previous study [167], the Tyr118 amino acid and HEM601 protein were described as essential residues, and in this study, the synthesized active compounds interacted significantly with Tyr118, His377, and HEM601 residues. The interactions with HEM were seen as π−π stacking and π−cation interactions. Therefore, the antifungal effects of compounds 113b, 113i, and 113j were considered to be caused by the destruction of cell integrity due to the inhibition of the LDM enzyme. The authors identified compound 6i with higher inhibitory activity due to H-bonding with Tyr132, unlike the other two compounds. Aryal et al. reported synthesis of 2-substituted benzimidazole containing 1,2,4-triazoles 114a and 114b (Figure 20). The compounds did not show antimicrobial activity against the tested strains Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis ATCC 1228 [168]. Kazeminejad et al. did a study on 1,2,4-triazoles as well as structure–activity relationships (SAR) [38].

Nandha et al. reported synthesis of 6-substituted-benzimidazoles with 1-(1,2,4-triazole) 115a–115d in three steps from 5-chloro-4-fluoro-2-nitrobenzenamine (Scheme 30). All compounds were screened against M. tuberculosis and four fungal strains, C. albicans, C. glabrata, C. krusei, and C. tropicalis. Compound 115c was the most active against M. tuberculosis and all tested fungal strains (MIC = 25 μg mL⁻¹) [169].